Evidence for Sigma Factor Competition in the Regulation of ...

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Evidence for Sigma Factor Competition in theRegulation of Alginate Production byPseudomonas aeruginosaYeshi YinMarshall University, [email protected]

T. Ryan WithersMarshall University, [email protected]

Xin Wang

Hongwei D. YuMarshall University, [email protected]

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Recommended CitationYin Y, Withers TR, Wang X, Yu HD (2013) Evidence for Sigma Factor Competition in the Regulation of Alginate Production byPseudomonas aeruginosa. PLoS ONE 8(8): e72329. doi:10.1371/journal.pone.0072329

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Evidence for Sigma Factor Competition in the Regulationof Alginate Production by Pseudomonas aeruginosaYeshi Yin1,3, T. Ryan Withers1, Xin Wang3, Hongwei D. Yu1,2,4*

1 Department of Biochemistry and Microbiology, Joan C. Edwards School of Medicine at Marshall University, Huntington, West Virginia, United States of America,

2 Department of Pediatrics, Joan C. Edwards School of Medicine at Marshall University, Huntington, West Virginia, United States of America, 3 Institute of Plant Protection

and Microbiology, Zhejiang Academy of Agricultural Sciences, Hangzhou, China, 4 Progenesis Technologies, LLC, Huntington, West Virginia, United States of America

Abstract

Alginate overproduction, or mucoidy, plays an important role in the pathogenesis of P. aeruginosa lung infection in cysticfibrosis (CF). Mucoid strains with mucA mutations predominantly populate in chronically-infected patients. However, themucoid strains can revert to nonmucoidy in vitro through suppressor mutations. We screened a mariner transposon libraryusing CF149, a non-mucoid clinical isolate with a misssense mutation in algU (AlgUA61V). The wild type AlgU is a stress-related sigma factor that activates transcription of alginate biosynthesis. Three mucoid mutants were identified withtransposon insertions that caused 1) an overexpression of AlgUA61V, 2) an overexpression of the stringent starvation proteinA (SspA), and 3) a reduced expression of the major sigma factor RpoD (s70). Induction of AlgUA61V in trans causedconversion to mucoidy in CF149 and PAO1DalgU, suggesting that AlgUA61V is functional in activating alginate production.Furthermore, the level of AlgUA61V was increased in all three mutants relative to CF149. However, compared to the wild typeAlgU, AlgUA61V had a reduced activity in promoting alginate production in PAO1DalgU. SspA and three other anti-s70

orthologues, P. aeruginosa AlgQ, E. coli Rsd, and T4 phage AsiA, all induced mucoidy, suggesting that reducing activity ofRpoD is linked to mucoid conversion in CF149. Conversely, RpoD overexpression resulted in suppression of mucoidy in allmucoid strains tested, indicating that sigma factor competition can regulate mucoidy. Additionally, an RpoD-dependentpromoter (PssrA) was more active in non-mucoid strains than in isogenic mucoid variants. Altogether, our results indicatethat the anti-s70 factors can induce conversion to mucoidy in P. aeruginosa CF149 with algU-suppressor mutation viamodulation of RpoD.

Citation: Yin Y, Withers TR, Wang X, Yu HD (2013) Evidence for Sigma Factor Competition in the Regulation of Alginate Production by Pseudomonasaeruginosa. PLoS ONE 8(8): e72329. doi:10.1371/journal.pone.0072329

Editor: Deepak Kaushal, Tulane University, United States of America

Received February 5, 2013; Accepted July 8, 2013; Published August 22, 2013

Copyright: � 2013 Yin et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricteduse, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: This work was supported by the National Aeronautics and Space Administration West Virginia Space Grant Consortium (NASA WVSGC) and the CysticFibrosis Foundation (CFF-YU11G0). H.D.Y. was supported by National Institutes of Health P20RR016477 and P20GM103434 to the West Virginia IDeA Network forBiomedical Research Excellence. T.R.W. was supported through the NASA WVSGC Graduate Research Fellowship. The funders had no role in study design, datacollection and analysis, decision to publish, or preparation of the manuscript.

Competing Interests: The authors declare that Hongwei D. Yu is the co-founder and Chief Science Officer of Progenesis Technologies, LLC (www.progenesistech.com), a biotech start-up company located in Huntington, West Virginia. Progenesis Technologies develops the recombinant alginate using thegenetically modified bacteria as the sources. There is one issued patent (U.S. Patent No. 7,781,166) and one pending patent (application No. 61/048,858) thatcover the technologies. Up till now, H.D.Y. hasn’t received financial compensation from Progeneis Technologies. Publication of this paper does not alter theauthors’ adherence to all the PLOS ONE policies on sharing data and materials.

* E-mail: [email protected]

Introduction

The Gram-negative bacterium P. aeruginosa is an important

opportunistic pathogen in humans, and has the potential to

proliferate in a wide range of niches. P. aeruginosa is one of the

major etiological agents of hospital-acquired infections and

ventilator-associated pneumonia [1]. More importantly, P. aerugi-

nosa is the leading cause of morbidity and mortality in cystic

fibrosis (CF) patients [2].

P. aeruginosa can produce a capsule-like polysaccharide called

alginate. Overproduction of alginate is also known as mucoidy [3].

Mucoid conversion facilitates the establishment of persistent

infection with P. aeruginosa in CF. The role of alginate in

pathogenesis includes: increased resistance to antibiotics [2],

increased resistance to phagocytic killing [4,5] and evasion of

the host’s immune response [4]. However, the mucoid phenotype

observed in CF isolates is extremely unstable ex vivo [6,7,8].

Reversion to non-mucoidy is common in vitro in the absence of a

selective pressure, and in vivo during the end-stage of CF disease

[9]. Although, environmental signals such as high osmolarity,

nitrogen or phosphate starvation, and ethanol-induced membrane

perturbation can activate transcription of algD encoding the key

enzyme for alginate biosynthesis [10], the selective pressure for

mucoid conversion of P. aeruginosa in CF respiratory environment

is not fully understood.

Several genes in P. aeruginosa are known to regulate alginate

production. Specifically, AlgU (AlgT, s22) is an alternative sigma

factor that drives the transcription of algD [11]; MucA is a trans-

membrane protein that negatively regulates alginate production by

sequestering AlgU [12]; MucB and proteases AlgW, MucP and

ClpXP affect alginate production by altering the stability of MucA

[13]. Mutations in mucA are recognized as the primary reason for

mucoid conversion in CF isolates [14,15,16,17]. However, the

reversion from a mucoid to a non-mucoid phenotype is still

possible. Sautter et al. isolated 34 spontaneous non-mucoid

variants from a MucA truncated mucoid stain PDO300 [18]. In

PLOS ONE | www.plosone.org 1 August 2013 | Volume 8 | Issue 8 | e72329

another study, 70% of the non-mucoid CF isolates carried a mucA

mutation [19].

The aim of this study was to better understand the alginate

regulation by determining if there are upstream mutations that

restore alginate overproduction to a clinical nonmucoid, algU mucA

double mutant (CF149). To achieve this objective, we conjugated

the mariner transposon plasmid pFAC [20] into the mucA mutant

CF149 with an alginate-suppressing mutation [21], and screened

for mucoid variants. Three genes were identified that regulated

mucoidy in CF149. Mechanistic studies suggest that mucoid

conversion in MucA truncated strains including CF149, is related

to competition between the two sigma factors, the major house

keeping sigma factor RpoD and AlgU for binding to the core RNA

polymerase (RNAP). Additionally, we documented that anti-s70

factors have the ability to induce mucoidy in the suppressed non-

mucoid P. aeruginosa strain CF149.

Materials and Methods

Bacteria Strains, Plasmids, and Growth ConditionsBacterial strains and plasmids used in this study are shown in

Table S1. Escherichia coli strains were grown at 37uC in Lennox

broth (LB) or LB agar. P. aeruginosa strains were grown at 37uC in

LB or on Pseudomonas isolation agar (PIA) plates (Difco). When

required, carbenicillin, tetracycline or gentamicin were added to

the broth or plates. The concentrations of carbenicillin, tetracy-

cline or gentamicin added in LB broth or plates were 100 mg ml21,

20 mg ml21 and 15 mg ml21, respectively. The concentration of

these antibiotics added to PIA plates were 300 mg ml21, 150 mg

ml21 or 300 mg ml21, respectively.

Figure 1. Increased alginate production in three mutants of CF149 with an algU-suppressor mutation. (A) Schematic diagram showingthe transposon insertions of CF149 (+algU), CF149 (+sspA), and CF149 (2rpoD), respectively. (B) Alginate production of CF149 (+algU), CF149 (+sspA),and CF149 (2rpoD) in comparison to other strains of P. aeruginosa. Three mucoid mutants were identified as a result of a transposon library screen.Alginate production was measured on PIA plates after incubation at 37uC for 24 hrs. Alginate production (mg/ml/OD600) was measured as describedin Materials and Methods. M and NM, represent mucoidy and nonmucoidy, respectively.doi:10.1371/journal.pone.0072329.g001

Alginate Regulation by Anti-s70 Factors


Phage Culture and Genomic DNA ExtractionE. coli BB was inoculated into 6 ml LB, and incubated at 37uC

with shaking. At an OD600 of 0.6, 200 ml of T4 phage stock was

added to the culture, and incubated overnight. Phage lysates were

collected using a 0.2 mm filter. Phage genomic DNA was extracted

using the standard procedure with phenol:chloroform:isoamyl

alcohol, and precipitated with ethanol.

Transformation and ConjugationThe One ShotH TOP10 (Invitrogen) chemical transformation

method was used according to the supplier’s instruction. The

transfer of plasmids from E. coli to Pseudomonas was performed via

triparental conjugations using the helper plasmid pRK2013 [22].

Transposon MutagenesisBiparental conjugations were carried out for transposon

mutagenesis, using E. coli SM10 lpir carrying plasmid pFAC as

the donor strain [20] and CF149 as the recipient strain. After

incubation, bacteria were collected and streaked onto PIA plates

supplemented with gentamicin (300 mg ml21). Mucoid colonies

were identified and subjected to further genetic analyses. The

chromosomal DNA of mucoid mutants was isolated using the

QIAamp genomic DNA Extraction kit (Qiagen). Approximately,

2 mg DNA was digested with SalI overnight at 37uC followed by

purification and self-ligation using Fast-Link DNA ligase (Epicen-

tre). The circular closed DNA was used as template for inverse

PCR using GM3OUT and GM5OUT primers [23]. The PCR

products were purified and sequenced. Finally, southern blot

hybridization was used to monitor the copy number of transposon

insertions using the GmR gene as the probe [24].

Protein Preparation, SDS-PAGE and Western BlottingBacteria were cultured on PIA plates for 24 hrs and then

collected for cell lysis. Following sonication, the protein concen-

tration of the resulting supernatant was measured using the Bio-

Rad Dc protein assay reagents (Bio-Rad). Equal amounts protein

were mixed with 26sample loading buffer and separated on a pre-

cast SDS-PAGE gels (Bio-Rad); Total proteins were transferred to

PVDF membrane (GE) for immuno-detection. A primary

monoclonal antibody of rat anti-HA (Roche) was used at a

dilution of 1:5000, while a goat anti-rat immunoglobulin G (heavy

and light chains) conjugated with horseradish peroxidase (Pierce)

(1:5000) was used as the secondary antibody. The immunoreactive

proteins were visualized using the Amersham ECL kit (GE).

Alginate AssayP. aeruginosa strains were grown at 37uC on triplicate PIA plates

for 24 hrs. The bacteria were collected and suspended in PBS.

The OD600 of bacterial suspension in PBS, which corresponds to

the bacterial density, was measured. The amount of uronic acid

was analyzed in comparison with a standard curve made with D-

mannuronic acid lactone (Sigma-Aldrich), as previously described

[25].

b-galactosidase Activity AssayPseudomonas strains carrying the plasmid pLP170 containing the

PssrA, PalgD and PalgW promoters fused with the promoterless lacZ

were cultured on three PIA plates. After 24 hrs, bacterial cells

were harvested and re-suspended in PBS. OD600 was measured

and adjusted to approximately 0.3. Cells were then permeabilized

using toluene, and the b-galactosidase activity was measured at

OD420 and OD550. The results of Miller Units were calculated

according to this formula: Miller Units = 10006[OD420–

(1.756OD550)]/[Reaction time (minutes)6Volume (ml)6OD600]

[26]. The reported values represent an average of three

independent experiments with standard error.

RNA Isolation and Real-time PCRBacteria total RNA were extracted with a RNeasy Mini Kit

(QIAGEN, USA) according to the manufacturer’s instructions.

The real-time PCR assays were performed on ABI PRISMH 7000

(ABI, USA) with One Step SYBRH PrimeScriptTM RT-PCR KIT

II (TaKaRa, Japan) according to the manufacturer’s instructions.

The rpoD gene was amplified using primers rpoD-RT-F (59-AGA

AGG ACG ACG AGG AAG A-39) and rpoD-RT-R (59-GCA

CCA GCT TGA TCG GCA TGA -39). The 16S rRNA gene was

amplified using primers UniF340 (59-ACT CCT ACG GGG

AGG CAG CAG T-39) and UniR514 (59-ATT ACC GCG GCT

GCT GGC-39) [27]. The relative expression level of rpoD was

Figure 2. Over-expression of SspA induces mucoidy in CF149.The sspA and sspB genes from P. aeruginosa were cloned behind thePBAD promoter in the pHERD20T vector, and conjugated into CF149. (A)Western blot analysis of SspA and SspB proteins using an anti-HAmonoclonal antibody. Lanes 1, 2 and 3 represent total cellular proteinsextracted from CF149 carrying pHERD20T-sspA, pHERD20T-sspB, andpHERD20T, respectively. (B) Morphology of CF149 containingpHERD20T-sspA, pHERD20T-sspB and pHERD20T. These strains werestreaked on PIA plates supplemented with 300 mg/ml of carbenicillin,0.1% L-Ara and incubated overnight at 37uC. Alginate production (mg/ml/OD600) was measured as described in Materials and Methods.doi:10.1371/journal.pone.0072329.g002



normalized to 16S rRNA, and calculated according to the

formula: fold change = 22DDct [28].

Measurement of Bacterial GrowthThe Pseudomonas strains were grown in LB culture medium

overnight at 37uC, then diluted to OD600 = 0.5 using PBS. Then

1 ml bacteria suspension was inoculated into a 250 ml flask

containing 50 ml Pseudomonas Isolation Broth (PIB; Alpha Biosci-

ences). To measure bacterial growth, OD600 was monitored every

2 hrs for 24 hrs. Triplicate of samples at each time point were

measured, and the means with standard error were used to

generate the growth curve.

Statistical AnalysisThe student t test and one-way ANOVA were performed using

the statistical software SPSS 13.0 (IBM, US) with P,0.05

considered as significant.

Results

Identification of Alginate Regulators in a P. aeruginosaStrain with a Suppressor Mutation

CF149 is a clinical isolate from a patient with CF [24]. CF149

displays a nonmucoid phenotype on PIA and PIA plus ammonium

metavanadate (PIA-AMV) plates [29]. Previously, we reported

CF149 have two mutations resulting in abrogation of an AlgU-

dependent transcription of lipotoxin LptF [21]. First, a frameshift

Table 1. The effect of anti-s70 factors on mucoid induction in strains of P. aeruginosa.

Strains MucA length AlgU length rsd (TOP 10) algQ b (PAO1) sspA (PAO1) asiA (T4 phage)

CF149 125+3 aa a Ala61Val (193 aa) M (29.0462.44) M (28.3362.74) M (51.2860.85) M (54.5162.28)

CF4349 125+3 aa WT (193 aa) NM (2.3560.18) NM (9.4961.26) NM (8.5362.06) NM (5.6062.07)

CF28 117 aa Tyr29Cys (193 aa) (193aa) NM (6.3762.51) NM (8.1062.79) NM (13.2060.72)(13.2060.72)(13.2060.72)

NM (11.5662.05)

CF17 143+3 aa WT (193 aa) NM (11.4562.97) NM (13.5262.64 NM (10.5760.75) NM (7.9460.72)

FRD2 143+3 aa Asp18Gly (193 aa) NM (14.0860.21) NM (11.7461.53) NM (7.0560.03) NM (14.6460.94)

PAO1 WT (194 aa) WT (193 aa) NM (14.4861.56) NM (7.8461.67) NM (9.3860.3) NM (17.7661.46)

PA14 WT (194 aa) WT (193 aa) NM (9.6361.25) NM (8.9561.15) NM (8.7560.24) NM (14.4762.59)

CF3715 WT (194 aa) WT (193 aa) NM (13.0563.60) NM (6.1060.67) NM (6.2060.19) NM (15.5961.28)

CF4009 WT (194 aa) WT (193 aa) NM (9.2160.49) NM (7.2462.31) NM (8.6960.11) NM (15.6462.52)

M and NM represent a mucoid and a non-mucoid phenotype, respectively, after incubation on PIA plates supplemented with 300 mg/ml of carbenicillin and 0.1% L-Araat 37uC for 24 hrs. The quantity of alginate production was measured (mean 6 standard error, mg/ml/OD600) and listed in the brackets.athe frameshift mutation in mucA results in the fusion of a truncated MucA (125 amino acids of N-terminal MucA) with an additional 3 amino acids with no homology tothe amino acid sequence of wild type MucA.ba concentration of 0.5% L-Ara was needed to induce the conversion to mucoidy in CF149. The alginate production of all AlgQ-overexpressing strains was measured onPIA plates containing 300 mg/ml of carbenicillin and 0.5% L-Ara.doi:10.1371/journal.pone.0072329.t001

Table 2. Over-expression of RpoD suppresses mucoidy in laboratory and clinical strains of P. aeruginosa.

StrainsAlginate production(mg/ml/OD600) MucA length AlgU length Alginate regulator

PHERD 20T-HA-rpoD-His

Laboratory strains PAO1-VE2 M (64.8066.52) WT (194 aa) WT (193 aa) Over-expression of MucE NMa

PAO1-VE13 M (20.9361.05) WT (194 aa) WT (193 aa) Inactivation of KinB NM

PAO1-VE19 M (61.2362.90) WT (194 aa) WT (193 aa) Inactivation of MucD NMa

PAO581 M (58.9563.07) 59 aa+35 aa WT (193 aa) MucA25 NMa

PAO579 M (48.0460.11) WT (194 aa) WT (193 aa) PilA108 NMa

Clinical strains CF149 (2rpoD) M (30.1660.61) 125+3 aa Ala61Val Reduced-expression of rpoD NM

CF149 (+algU) M (44.2060.49) 125+3 aa Ala61Val Over-expression of algU NMa

CF149 (+sspA) M (25.4061.07) 125+3 aa Ala61Val Over-expression of sspA NM

FRD1 M (56.2764.06) 143+3 aa WT MucA22 NM

PDO300 M (68.0861.25) 143+3 aa WT MucA22 NM

CF1003M M (30.6162.28) 59 aa+35 aa WT (193 aa) Unknown NM

CF7447M M (20.2261.40) WT (194 aa) WT (193 aa) Unknown NM

NM and M represent non-mucoidy and mucoidy, respectively, after incubation on PIA plates supplemented with 300 mg/ml of carbenicillin and 0.1% L-Ara at 37uC for24 hrs.aa concentration of 0.5% L-Ara was needed to completely suppress mucoidy. The values for alginate production represent an average of three independentexperiments with standard error.doi:10.1371/journal.pone.0072329.t002



Figure 3. Alginate induction by CF149 AlgUA61V, the detection of AlgUA61V and the promoter activity of PalgD-lacZ in CF149 (+algU),CF149 (+sspA) and CF149 (2rpoD). (A) AlgUA61V retained the function of inducing alginate production. The wild type algU gene of PAO1 and itsvariant of CF149 were cloned into pHERD20T, and conjugated into PAO1DalgU. Strains containing pHERD20T-algUWT (PAO1), pHERD20T-algUA61V

(CF149), and pHERD20T were streaked on PIA plates supplemented with 300 mg/ml of carbenicillin and incubated overnight at 37uC. Alginateproduction (mg/ml/OD600) was measured as described in Materials and Methods. (B) The level of AlgU in CF149, CF149 (+algU), CF149 (2rpoD) andCF149 (+sspA) was detected using Western blot with anti-AlgU monoclonal antibody [16]. (C) Measurement of the activity of the algD promoter in therespective strains. The PalgD promoter was inserted into pLP170 vector containing the promoterless lacZ gene. The pLP170 PalgD-lacZ was transferredinto the respective strains via triparental conjugation. The b-galactosidase activity was measured as described in Materials and Methods. *, representsa significant difference compared to CF149 (P,0.05).doi:10.1371/journal.pone.0072329.g003



mutation in mucA is predicted to produce a truncated MucA

protein with 128 amino acids in contrast to wild type MucA with

194 amino acids. Second, CF149 carries a missense mutation in

algU predicted to result in a substitution of alanine for valine at

position 61 of the primary amino acid sequence of AlgU

(AlgUA61V) [21]. To determine whether the alginate suppressor

strain CF149 still had the ability to restore mucoidy, a transposon

library was constructed and screened. As seen in Figure 1A, we

identified three insertions that promote alginate production [17].

Southern blot analysis showed that only one copy of the

transposon was inserted on the chromosome in these mucoid

strains (data not shown). We mapped the insertion site using

inverse PCR as previously described [17,23]. Two insertions were

identified in intergenic regions between PA0762 (algU) and

PA0761 (nadB), and PA4428 (sspA) and PA4429 (probable

cytochrome c1 precursor) (Figure 1A). In these two mutants, the

transposon was upstream of algU and sspA, and was oriented in the

same direction as the previously-observed insertion causing an

overexpression of mucE [17]. These two mutants likely had an

overexpression of algU and sspA from read through of the

gentamicin-resistance gene (aacC1) promoter (PGm) [30]. These

strains were named CF149 (+algU) and CF149 (+sspA). The third

mutant had an insertion site of 78 base pairs behind the stop

codon of the rpoD gene (Figure 1A). The rpoD gene (PA0576)

encodes the major housekeeping sigma factor (s70). RpoD is

essential for the growth and viability of cells, and no rpoD mutant

has been reported in the P. aeruginosa mutant bank [31]. Because

the orientation of the pFAC PGm is opposite to the direction of

rpoD gene, the insertion is predicted to reduce the expression of

rpoD. This strain was named CF149 (2rpoD). To verify the

decreased transcription of rpoD in CF149 (2rpoD), we compared

the transcript level by real-time PCR, and the activity of an RpoD-

dependent promoter PssrA-lacZ in two isogenic strains. The results

showed that the transcript level of rpoD in CF149 (2rpoD) was 60%

of the value of CF149. The Miller assay results also showed that

the PssrA-lacZ activity reduced by 68% in CF149 (2rpoD) compared

to CF149. The alginate production by these mucoid strains was

also measured and shown to be more than 2-fold higher than the

parent strain (Figure 1B).

SspA, not SspB, Induces Mucoidy in CF149The operon of sspAB encodes the stringent starvation proteins A

and B that function in response to amino acid starvation [32]. The

sspA and sspB genes share the same promoter, and are co-

expressed in E. coli [32]. SspB is also a specificity-enhancing factor

for the protease ClpXP in E. coli [33]. ClpXP has been reported to

regulate alginate production in P. aeruginosa [23]. In order to

determine which gene is responsible for the mucoid conversion in

CF149 (+sspA), P. aeruginosa sspA and sspB were cloned behind the

PBAD promoter in the shuttle vector pHERD20T [34]. As seen in

Figure 2A, we detected the expression of SspA and SspB in CF149

after induction with 0.1% L-arabinose (L-Ara). However, we

observed a mucoid phenotype only with the overexpression of sspA

(Figure 2B), indicating that SspA is an inducer of alginate

production when overexpressed in CF149.

Anti-s70 Factors Induce Conversion to Mucoidy in CF149The mucoid phenotype expressed by CF149 (+sspA) and CF149

(2rpoD) suggests that RpoD may be involved in the alginate

regulation. Schlictman et al. reported that E. coli sspA can

complement the algQ mutation by restoring mucoidy in a CF

clinical isolate [35]. AlgQ and Rsd belong to the family of the

regulators of the major sigma factor RpoD (s70) in Proteobacteria.

Members of the anti-s70 factors are thought to interact with the

conserved region 4 of s70 subunit of RNAP [36]. Similarly, AsiA,

encoded in the T4 phage, also functions as an anti-s70 factor [37].

Figure 4. The effect of mucA on the mucoid suppression in CF149 (+algU), CF149 (+sspA) and CF149 (2rpoD). AlgUA61V–inducedmucoidy was suppressed by the wild type mucA in trans. The mucA gene was over-expressed in trans in CF149(+algU), CF149(2rpoD) andCF149(+sspA) by adding 0.1% L-Ara in PIA supplemented with 300 mg/ml carbenicllin. The plate was incubated for 24 hrs at 37uC.doi:10.1371/journal.pone.0072329.g004



Our hypothesis is that CF149 (+sspA) and CF149 (2rpoD) utilize

the mechanism of modulating RpoD to become mucoid. To test

this, we cloned the rsd gene from E.coli, algQ and sspA from P.

aeruginosa, and asiA from T4 phage in pHERD20T. As seen in

Table 1, overexpression of these genes caused conversion to

mucoidy in CF149. However, the anti-s70 factors had no effect on

mucoid induction in other strains tested, even though CF4349 has

a wild type algU and the same predicted length of MucA as CF149

(Table 1). However, mucoidy of CF149 (+sspA) could be due to a

non-specific effect. To test this possibility, we examined the effect

of anti-RpoF factor FlgM on mucoid conversion. As seen in Figure

S1, FlgM had no effect on mucoid induction in PAO1 and CF149.

Overexpression of rpoD Results in Suppression ofMucoidy

Mucoidy in CF149(2rpoD), CF149(+sspA) and CF149(+algU )

could be due to the competition between RpoD and AlgU for

RNAP. To test this hypothesis, rpoD was cloned and overexpressed

in various mucoid strains. The mucoid laboratory and clinical

isolates were all suppressed by the overexpression of RpoD

(Table 2). However PAO1-VE2, PAO1-VE19, PAO579 and

PAO581 required a higher concentration of L-ara (0.5% vs. 0.1%)

for a complete suppression of mucoidy (Table 2). To further test

the hypothesis that sigma factor competition can reduce the

activity of AlgU resulting in the suppression of alginate production,

we induced RpoN (s54), RpoS(s38), and RpoF(s28) in various

Figure 5. Regulation of RpoD- and AlgU-dependent promoters in isogenic non-mucoid and mucoid strains of P. aeruginosa. (A) The b-galactosidase activity of AlgU-dependent promoter PalgD-lacZ and alginate production (mg/ml/OD600) were measured in non-mucoid and mucoidstrains. (B) The b-galactosidase activity of an RpoD-dependent promoter PssrA-lacZ was measured in non-mucoid and mucoid strains. *, represents asignificant difference compared to PAO1 (P,0.05).doi:10.1371/journal.pone.0072329.g005



mucoid mutants. As expected, overexpression of these sigma

factors suppressed alginate production (Table S2). We also

observed that overexpressed RpoD was unstable in P. aeruginosa

and E.coli (Figure S2).

Missense Mutation in CF149 algU Results in Productionof a Variant of AlgU with Reduced Function

One explanation for mucoidy in CF149 (+algU) is that AlgUA61V

retains some function despite the amino acid substitution. To test

this, we compared the function of AlgUA61V vs. wild type AlgU by

measuring the amount of alginate induced by two forms of AlgU

in the PAO1DalgU strain. CF149 AlgUA61V kept the ability to

induce mucoidy, albeit with a reduced amount of alginate

(Figure 3A). To explain how the three mutants become mucoid,

we next measured the level of AlgUA61V in the total cell lysates of

CF149 (+algU), CF149 (2rpoD) and CF149 (+sspA) through

Western blot. The AlgU protein level was increased in CF149

(+algU), CF149 (2rpoD) and CF149 (+sspA) compared to the parent

CF149 (Figure 3B). The promoter activity of PalgD-lacZ also

increased in these mucoid strains (Figure 3C).We also tested the

hypothesis that the mucoidy in all three mutants was due to the

increased expression of AlgUA61V. To do so, we introduced

pHERD20T-AlgUA61V into CF149. As predicted, CF149 carrying

pHERD20T-AlgUA61V displayed a mucoid phenotype (data not

shown). Furthermore, the absence of AlgUA61V in Figure 3B is

consistent with non-mucoidy of CF149 on PIA, which is probably

due to the reduced auto-regulatory activity of AlgUA61V [38].

Intramembrane Proteolysis has a Minimal Role in theRegulation of Mucoidy in CF149

In wild type strain PAO1, AlgU can be sequestered by wild type

MucA, thereby preventing it from activating the alginate

biosynthetic operon [12,38,39]. Since all three mutants of

CF149 have an increased level of AlgUA61V, we next investigated

whether the wild type MucA can still exert an inhibitory effect on

AlgUA61V. The wild type mucA gene was transferred into these

mucoid strains: CF149 (+algU), CF149 (+sspA) and CF149 (2rpoD).

Over-expression of mucA suppressed mucoidy (Figure 4) suggesting

that the mucoidy of all three mutants is due to the activation of the

AlgU pathway. But the mutant MucA in CF149 has 128 amino

Figure 6. Mucoid mutants of P. aeruginosa display a reduced growth rate compared to the isogenic nonmucoid strain PAO1. Thegrowth curves were created by growth of PAO1 (nonmucoid), VE2 (mucoid) and PAO581 (mucoid) in PIB. The horizontal axis represents time in hrs,while the vertical axis is the optical density at 600 nm. *, represents a significant difference compared to PAO1 (P,0.05).doi:10.1371/journal.pone.0072329.g006

Figure 7. Schematic diagram for mucoid conversion in CF149caused by competition between two sigma factors, RpoD andAlgU. Stress or starvation can affect the expression of anti-s70 factorsresulting in a decrease in the activity of RpoD, which in turn increasesthe chances for the alternative sigma factor AlgU to bind to the coreRNAP to initiate transcription for alginate biosynthesis. MucA is an anti-sigma factor that has the ability to inhibit the activity of AlgU in alginateproduction. Competition for RNAP between RpoD and AlgU candetermine which promoter is activated. RpoD binds to core RNAP topromote the transcription of housekeeping genes, while AlgU binds tocore RNAP at the PalgD promoter to activate alginate production.doi:10.1371/journal.pone.0072329.g007



acid residues, and carries the intact trans-membrane domain of

MucA84–104. We also noticed that the promoter activity of PalgW in

CF149 (2rpoD) was increased compared to CF149 (Figure S3). To

test if intramembrane proteolysis has a role in cleaving the

periplasmic portion of MucA in CF149, we overexpressed

proteases algW, mucP, clpX, clpP and clpP2 and found this had no

effect on mucoid induction in CF149. Together, these results

suggest that these proteases have a minimal role in regulating the

mucoid conversion in CF149, or they may require a mechanism of

activation which is absent in CF149.

Competition between RpoD- and AlgU-dependentPromoters in Mucoid and Non-mucoid Strains

Since the transcription from two promoters, PalgD and PssrA is

AlgU-dependent and RpoD-dependent, respectively, we cloned

these two promoters into a lacZ fusion vector (pLP170) and the

reporter b-galactosidase activity was measured [26]. As seen in

Figure 5A, alginate production in the mucoid strains VE2 and

PAO581 was higher than that in the non-mucoid strain PAO1.

Similarly, the promoter activity of PalgD also was higher in these

mucoid strains (Figure 5A). However, the promoter activity of PssrA

was inversely related to PalgD activity and alginate production

(Figure 5B), suggesting that the RpoD-dependent promoter was

less active in mucoid cells than in the isogenic non-mucoid cells.

We also measured the growth curve for mucoid and non mucoid

strains. As seen in Figure 6, the growth rate for mucoid strains was

reduced after 16 hrs growth for VE2 and PAO581 in comparison

with nonmoucid strain PAO1.

Discussion

Individuals with CF are thought to acquire initial colonization

of P. aeruginosa from environmental sources [9]. These early

colonizing strains display a non-mucoid phenotype with a wild-

type MucA [17]. Due to strong selective pressure in CF lungs,

mucoid mucA mutants eventually become a dominant population

[14,40]. However, secondary mutations that suppress alginate

overproduction have been reported [8,19]. One presumed

advantage with non-mucoid suppressors is the loss of mucoid

status is coupled with the presence of the flagella, which may

promote the colonization of new niches in the lungs [41,42].

Through screening a transposon library, we found that overex-

pression of sspA and CF149 algU, and reduced expression of rpoD,

are functionally equivalent in causing mucoid conversion in the

non-mucoid clinical isolate CF149. This mucoid phenotype can be

suppressed by overexpression of the anti-sigma factor MucA [12].

We propose that the mechanism for mucoid conversion mediated

by AlgU, SspA and RpoD in CF149 may be related to the

competition between sigma factors RpoD and AlgU for the core

RNAP binding site (Figure 7). Because of the differential binding

ability among sigma factors for core RNAP [43], s factor

competition exists within a cell at any given time [44]. We

investigated whether this competition is also present in CF149 and

CF149(2rpoD), by measuring the promoter activity of Palgw whose

activation depends on RpoN [45], and PalgD which is driven by

AlgU [11]. As seen in Figures 3C and S3, activities of both PalgD

and PalgW were increased in CF149 (2rpoD). Furthermore, the

mucoid suppression resulting from the overexpression of RpoD

can be attributed to the competition between two sigma factors

(Table 2). Table S2 illustrates that sigma factors besides RpoD can

also exert the same effect on mucoid suppression in mucA plus and

minus mucoid strains. Thus, any major shift in the intracellular

level of sigma factors can potentially affect mucoid conversion,

because the pool of core RNAP, which is made up of five different

subunits, must be in a limiting amount inside bacterial cells.

However, data in Figure S1 demonstrate that induction of FlgM,

which is the anti-sigma factor for RpoF responsible for the

transcription initiation of flagella biosynthesis [46], failed to induce

mucoidy in PAO1 and CF149. This may be due to the fact that

the impact on the pool of RNAP is somewhat different between a

minor sigma factor RpoF and a major sigma factor RpoD.

Therefore not all anti-sigma factors are functionally equal in terms

of alginate induction.

Anti-sigma factors are proteins that bind to cognate sigma

factors, thereby inhibiting their transcriptional activity [47]. The

E.coli protein Rsd associates specifically with s70 to inhibit the s70-

dependent transcription [48]. The P. aeruginosa transcriptional

regulator AlgQ/AlgR2, shares 55% identity at the amino acid

sequence level with Rsd [48], and has been proposed as an anti-

s70 factor that interacts with s70 to affect the transcriptional

activity of algD [49]. The T4 phage anti-s70 factor AsiA also has a

similar function as Rsd and AlgQ. Specifically, AsiA can regulate

the transcriptional activity of rpoD and other promoters

[37,50,51,52]. Although the amino acid sequence similarity is

low (,10%) between P. aeruginosa SspA to anti-s70 factors Rsd,

AsiA and AlgQ, the similarity at amino acid level between E. coli

SspA to P. aeruginosa SspA is higher than 50%. Hansen et al

reported that E. coli SspA is an RNAP-associated protein, and can

down-regulate expression from the s70-dependent promoters [53].

More importantly, E. coli SspA can functionally replace P.

aeruginosa anti-sigma factor AlgQ [35]. The sequence diversity in

anti-sigma factors suggests that they may have different binding

sites on RpoD [36]. Their inhibitory activity to the s70-driven

transcription is therefore different. Compared with the strong

inhibition of AsiA, Rsd shows only a modest effect on s70

transcription in vitro and in vivo [48,54,55]. This may explain why

overexpression of Rsd and AsiA can induce mucoidy in CF149

when the growth media are supplemented with 0.1% L-Ara, while

AlgQ-induced mucoidy in this strain requires a higher concentra-

tion of 0.5% L-Ara (Table 1). Also, compared with Rsd and AsiA,

AlgQ does not have the ability to significantly inhibit the growth of

E. coli in vivo [54].

In the current study, anti-s70 factors were found to have an

effect on mucoid induction in suppressed nonmucoid strain

CF149. This induction is not directly through the interaction

with the core RNAP, rather it is through the reduction of RpoD

activity. CF149 may be a rare alginate suppressor mutant. The

amino acid substitution in AlgUA61V in CF149 was mapped to the

conserved region of Sigma 70_r2 Superfamily [NCBI CDD

cl08419]. This region contains both the 210 promoter recognition

helix and the primary core RNAP binding determinant. However,

the change from alanine to valine is of conservative nature (non-

polar to non-polar), which may explain why this mutation renders

AlgUA61V partially defective. Therefore, it is not surprising to see

that not all clinical isolates respond to the induction by anti-s70

factors in the same manner (Table 1). For example, CF4349 is not

inducible, even though it has a wild type AlgU and the mucA

genotype is the same as CF149. This suggests that there could be

another unknown suppressor mutation, which nullifies the effect of

anti-s70 induction in CF4349, or the mutation in CF149

AlgUA61V somehow amplifies the effect of anti-s70 induction.

Earlier work showed that the growth condition that promotes

the fast growth rate of P. aeruginosa PAO1 does not select for the

mucoid variants, but the condition that caused a slow growth rate

does [56]. In Salmonella, the expression of RpoE, the AlgU

homolog induced the stationary phase of growth (slow growth rate)

[57]. In the current study, overexpression of anti-RpoD factors can

induce mucoidy in CF149 and overexrpression of RpoD can



inhibit mucoidy in all tested strains. Furthermore, there is a

competition between RpoD- and AlgU-dependent promoters

(Figure 5), and the growth rate of both mucoid strain VE2 and

PAO581 were slower than that of the isogenic nonmucoid strain

PAO1 (Figure 6). These data provide genetic evidence for the

sigma factor competiton in the regulation of alginate production

by P. aeruginosa.

Mucoid conversion in P. aeruginosa can be achieved through two

known mechanisms. Intra-membrane proteolysis acts as a primary

mechanism to initiate the degradation of MucA in P. aeruginosa

with wild type mucA [13]. Mutation of the mucA gene is another

mechanism to become mucoid and is prevalent in isolates from

chronic lung infections in CF [2]. In the current study, we showed

that a missense mutation in algU causes the loss of mucoidy in the

mucA mutant, but augmentation of anti-s70 factors such as SspA

leads to increased amounts of AlgU, causing mucoid conversion in

the AlgU-suppressed strain. Williams et al reported that carbon,

amino acid, nitrogen and phosphate starvation can induce the

expression of SspA [32], and Roychoudhury et al found that

starvation caused by nitrogen and phosphate limitation is one of

the signals leading to AlgQ-mediated activation of the algD

promoter in P. aeruginosa [58]. Therefore, stress signals may

activate the expression of anti-RpoD factors, thus causing mucoid

conversion in the suppressed strains.

In summary, we found three genes algU, sspA and rpoD that

regulate the conversion to mucoidy in the MucA-truncated, non-

mucoid P. aeruginosa strain CF149, which contains an AlgU-

suppressor mutation. Interestingly, our data indicate the missense

mutation in CF149 AlgU only reduces, but does not completely

abolish the function as the sigma factor that drives alginate

biosynthetic operon. The mechanism by which these genes cause

mucoidy may be due to competition between the sigma factors

AlgU and RpoD. Also, anti-s70 factors AsiA, Rsd, AlgQ and SspA

can induce mucoidy in strain CF149.

Acknowledgments

We thank Richard M. Niles for assistance in revising this

manuscript, and Gary Schultz from the Department of Biology at

Marshall University for providing the T4 bacteriophage and its

host E. coli BB.

Supporting Information

Figure S1 FlgM, an anti-sigma factor for RpoF, fails toinduce mucoidy in CF149 and PAO1. pHERD20T-flgM was

conjugated into CF149 and PAO1, respectively. Strains carrying

pHERD20T-flgM, pHERD20T-sspA, and pHERD20T were

incubated on PIA plates supplemented with 300 mg/ml carben-

icillin, 0.1% L-ara and incubated at 37uC for 24 hrs. Alginate was

harvested and measured as described in Materials and Methods. *,

represents a significant difference between each group (P,0.05).

(TIF)

Figure S2 The stability of over-expressed RpoD in P.aeruginosa and E.coli. RpoD was expressed in P. aeruginosa

PAO581 (A) and E. coli TOP10 cells (B) carrying pHERD20T-

HA-rpoD-His under the induction with different concentration of

L-Ara. PAO581 carried pHERD20T-HA-rpoD-His was cultured

on PIA plates supplemented with 300 mg/ml carbenicllin and

different concentrations of L-Ara for 24 hrs and the cells were then

collected for cell lysis. TOP10 carried pHERD20T-HA-rpoD-His

was cultured on LB plates supplemented with 100 mg/ml

carbenicllin and L-Ara for 24 hours. Following sonication, 50 mg

protein of total cell lysate from each sample was used for SDS-

PAGE and Western blotting analysis. Lane 1: the protein

molecular mass standards; Lane 2: cells no L-Ara; Lane 3: cells

induced by adding 0.1% L-Ara; Lane 4: cells induced by adding

0.5% L-Ara; Lane 5: control with the empty vector.

(TIF)

Figure S3 Reduced expression of RpoD inCF149(2rpoD) is correlated with increased promoteractivity of PalgW. RpoN dependent promoter pLP170-PalgW was

conjugated into CF149 and CF149 (2rpoD), respectively. The

Miller assay was used to detect the activation of PalgW in these

strains. *, represents the difference of the b-galactosidase activity

between these strains is significant (P,0.05).

(TIF)

Table S1 Strains and plasmids used in this study.

(DOC)

Table S2 Inhibitory effect of sigma factors RpoN, RpoSand RpoF on P. aeruginosa mucoidy.

(DOC)

Author Contributions

Conceived and designed the experiments: YY HDY. Performed the

experiments: YY TRW. Analyzed the data: YY TRW HDY. Wrote the

paper: YY TRW XW HDY.

References

1. Bodey GP, Bolivar R, Fainstein V, Jadeja L (1983) Infections caused by

Pseudomonas aeruginosa. Rev Infect Dis 5: 279–313.

2. Govan JR, Deretic V (1996) Microbial pathogenesis in cystic fibrosis: mucoid

Pseudomonas aeruginosa and Burkholderia cepacia. Microbiol Rev 60: 539–574.

3. Evans LR, Linker A (1973) Production and characterization of the slime

polysaccharide of Pseudomonas aeruginosa. J Bacteriol 116: 915–924.

4. Leid JG, Willson CJ, Shirtliff ME, Hassett DJ, Parsek MR, et al. (2005) The

exopolysaccharide alginate protects Pseudomonas aeruginosa biofilm bacteria from

IFN-gamma-mediated macrophage killing. J Immunol 175: 7512–7518.

5. Pier GB, Coleman F, Grout M, Franklin M, Ohman DE (2001) Role of alginate

O acetylation in resistance of mucoid Pseudomonas aeruginosa to opsonic

phagocytosis. Infect Immun 69: 1895–1901.

6. MacGeorge J, Korolik V, Morgan AF, Asche V, Holloway BW (1986) Transfer

of a chromosomal locus responsible for mucoid colony morphology in

Pseudomonas aeruginosa isolated from cystic fibrosis patients to P. aeruginosa PAO.

J Med Microbiol 21: 331–336.

7. Ohman DE, Chakrabarty AM (1981) Genetic mapping of chromosomal

determinants for the production of the exopolysaccharide alginate in a

Pseudomonas aeruginosa cystic fibrosis isolate. Infect Immun 33: 142–148.

8. Schurr MJ, Martin DW, Mudd MH, Deretic V (1994) Gene cluster controlling

conversion to alginate-overproducing phenotype in Pseudomonas aeruginosa:

functional analysis in a heterologous host and role in the instability of mucoidy.

J Bacteriol 176: 3375–3382.

9. Hogardt M, Heesemann J (2010) Adaptation of Pseudomonas aeruginosa during

persistence in the cystic fibrosis lung. Int J Med Microbiol 300: 557–562.

10. May TB, Shinabarger D, Maharaj R, Kato J, Chu L, et al. (1991) Alginate

synthesis by Pseudomonas aeruginosa: a key pathogenic factor in chronic pulmonary

infections of cystic fibrosis patients. Clin Microbiol Rev 4: 191–206.

11. Martin DW, Holloway BW, Deretic V (1993) Characterization of a locus

determining the mucoid status of Pseudomonas aeruginosa: AlgU shows sequence

similarities with a Bacillus sigma factor. J Bacteriol 175: 1153–1164.

12. Xie ZD, Hershberger CD, Shankar S, Ye RW, Chakrabarty AM (1996) Sigma

factor-anti-sigma factor interaction in alginate synthesis: inhibition of AlgT by

MucA. J Bacteriol 178: 4990–4996.

13. Damron FH, Goldberg JB (2012) Proteolytic regulation of alginate overpro-

duction in Pseudomonas aeruginosa. Mol Microbiol 84: 595–607.

14. Boucher JC, Yu H, Mudd MH, Deretic V (1997) Mucoid Pseudomonas aeruginosa

in cystic fibrosis: characterization of muc mutations in clinical isolates and

analysis of clearance in a mouse model of respiratory infection. Infect Immun 65:

3838–3846.

15. Mathee K, McPherson CJ, Ohman DE (1997) Posttranslational control of the

algT (algU)-encoded sigma22 for expression of the alginate regulon in Pseudomonas



aeruginosa and localization of its antagonist proteins MucA and MucB (AlgN).

J Bacteriol 179: 3711–3720.16. Schurr MJ, Yu H, Martinez-Salazar JM, Boucher JC, Deretic V (1996) Control

of AlgU, a member of the sigma E-like family of stress sigma factors, by the

negative regulators MucA and MucB and Pseudomonas aeruginosa conversion tomucoidy in cystic fibrosis. J Bacteriol 178: 4997–5004.

17. Qiu D, Eisinger VM, Rowen DW, Yu HD (2007) Regulated proteolysis controlsmucoid conversion in Pseudomonas aeruginosa. Proc Natl Acad Sci U S A 104:

8107–8112.

18. Sautter R, Ramos D, Schneper L, Ciofu O, Wassermann T, et al. (2012) Acomplex multilevel attack on Pseudomonas aeruginosa algT/U expression and algT/

U activity results in the loss of alginate production. Gene 498: 242–253.19. Ciofu O, Mandsberg LF, Bjarnsholt T, Wassermann T, Hoiby N (2010) Genetic

adaptation of Pseudomonas aeruginosa during chronic lung infection of patients withcystic fibrosis: strong and weak mutators with heterogeneous genetic back-

grounds emerge in mucA and/or lasR mutants. Microbiology 156: 1108–1119.

20. Wong SM, Mekalanos JJ (2000) Genetic footprinting with mariner-basedtransposition in Pseudomonas aeruginosa. Proc Natl Acad Sci U S A 97: 10191–

10196.21. Damron FH, Napper J, Teter MA, Yu HD (2009) Lipotoxin F of Pseudomonas

aeruginosa is an AlgU-dependent and alginate-independent outer membrane

protein involved in resistance to oxidative stress and adhesion to A549 humanlung epithelia. Microbiology 155: 1028–1038.

22. Figurski DH, Helinski DR (1979) Replication of an origin-containing derivativeof plasmid RK2 dependent on a plasmid function provided in trans. Proc Natl

Acad Sci U S A 76: 1648–1652.23. Qiu D, Eisinger VM, Head NE, Pier GB, Yu HD (2008) ClpXP proteases

positively regulate alginate overexpression and mucoid conversion in Pseudomonas

aeruginosa. Microbiology 154: 2119–2130.24. Head NE, Yu H (2004) Cross-sectional analysis of clinical and environmental

isolates of Pseudomonas aeruginosa: biofilm formation, virulence, and genomediversity. Infect Immun 72: 133–144.

25. Damron FH, Qiu D, Yu HD (2009) The Pseudomonas aeruginosa sensor kinase

KinB negatively controls alginate production through AlgW-dependent MucAproteolysis. J Bacteriol 191: 2285–2295.

26. Miller JH (1972) beta-galactosidase assay. In: Miller JH, editor. Experiments inmolecular genetics. Cold Spring Harbor, New York: Cold Spring Harbor

Laboratory. 352–355.27. Barman M, Unold D, Shifley K, Amir E, Hung K, et al. (2008) Enteric

salmonellosis disrupts the microbial ecology of the murine gastrointestinal tract.

Infect Immun 76: 907–915.28. Pfaffl MW (2001) A new mathematical model for relative quantification in real-

time RT-PCR. Nucleic Acids Res 29: e45.29. Damron FH, Davis MR, Jr., Withers TR, Ernst RK, Goldberg JB, et al. (2011)

Vanadate and triclosan synergistically induce alginate production by Pseudomonas

aeruginosa strain PAO1. Mol Microbiol 81: 554–570.30. Rubin EJ, Akerley BJ, Novik VN, Lampe DJ, Husson RN, et al. (1999) In vivo

transposition of mariner-based elements in enteric bacteria and mycobacteria.Proc Natl Acad Sci U S A 96: 1645–1650.

31. Jacobs MA, Alwood A, Thaipisuttikul I, Spencer D, Haugen E, et al. (2003)Comprehensive transposon mutant library of Pseudomonas aeruginosa. Proc Natl

Acad Sci U S A 100: 14339–14344.

32. Williams MD, Ouyang TX, Flickinger MC (1994) Starvation-inducedexpression of SspA and SspB: the effects of a null mutation in sspA on Escherichia

coli protein synthesis and survival during growth and prolonged starvation. MolMicrobiol 11: 1029–1043.

33. Levchenko I, Seidel M, Sauer RT, Baker TA (2000) A specificity-enhancing

factor for the ClpXP degradation machine. Science 289: 2354–2356.34. Qiu D, Damron FH, Mima T, Schweizer HP, Yu HD (2008) PBAD-based

shuttle vectors for functional analysis of toxic and highly regulated genes inPseudomonas and Burkholderia spp. and other bacteria. Appl Environ Microbiol 74:

7422–7426.

35. Schlictman D, Shankar S, Chakrabarty AM (1995) The Escherichia coli genes sspA

and rnk can functionally replace the Pseudomonas aeruginosa alginate regulatory

gene algR2. Mol Microbiol 16: 309–320.36. Yuan AH, Gregory BD, Sharp JS, McCleary KD, Dove SL, et al. (2008) Rsd

family proteins make simultaneous interactions with regions 2 and 4 of theprimary sigma factor. Mol Microbiol 70: 1136–1151.

37. Orsini G, Ouhammouch M, Le Caer JP, Brody EN (1993) The asiA gene of

bacteriophage T4 codes for the anti-sigma 70 protein. J Bacteriol 175: 85–93.38. Hershberger CD, Ye RW, Parsek MR, Xie ZD, Chakrabarty AM (1995) The

algT (algU) gene of Pseudomonas aeruginosa, a key regulator involved in alginate

biosynthesis, encodes an alternative sigma factor (sigma E). Proc Natl AcadSci U S A 92: 7941–7945.

39. Potvin E, Sanschagrin F, Levesque RC (2008) Sigma factors in Pseudomonas

aeruginosa. FEMS Microbiol Rev 32: 38–55.

40. Yoon SS, Coakley R, Lau GW, Lymar SV, Gaston B, et al. (2006) Anaerobic

killing of mucoid Pseudomonas aeruginosa by acidified nitrite derivatives under cysticfibrosis airway conditions. J Clin Invest 116: 436–446.

41. Garrett ES, Perlegas D, Wozniak DJ (1999) Negative control of flagellumsynthesis in Pseudomonas aeruginosa is modulated by the alternative sigma factor

AlgT (AlgU). J Bacteriol 181: 7401–7404.42. Tart AH, Blanks MJ, Wozniak DJ (2006) The AlgT-dependent transcriptional

regulator AmrZ (AlgZ) inhibits flagellum biosynthesis in mucoid, nonmotile

Pseudomonas aeruginosa cystic fibrosis isolates. J Bacteriol 188: 6483–6489.43. Maeda H, Fujita N, Ishihama A (2000) Competition among seven Escherichia coli

sigma subunits: relative binding affinities to the core RNA polymerase. NucleicAcids Res 28: 3497–3503.

44. Ishihama A (2000) Functional modulation of Escherichia coli RNA polymerase.

Annu Rev Microbiol 54: 499–518.45. Damron FH, Owings JP, Okkotsu Y, Varga JJ, Schurr JR, et al. (2012) Analysis

of the Pseudomonas aeruginosa regulon controlled by the sensor kinase KinB andsigma factor RpoN. J Bacteriol 194: 1317–1330.

46. Starnbach MN, Lory S (1992) The fliA (rpoF) gene of Pseudomonas aeruginosa

encodes an alternative sigma factor required for flagellin synthesis. Mol

Microbiol 6: 459–469.

47. Hughes KT, Mathee K (1998) The anti-sigma factors. Annu Rev Microbiol 52:231–286.

48. Jishage M, Ishihama A (1998) A stationary phase protein in Escherichia coli withbinding activity to the major sigma subunit of RNA polymerase. Proc Natl Acad

Sci U S A 95: 4953–4958.

49. Dove SL, Hochschild A (2001) Bacterial two-hybrid analysis of interactionsbetween region 4 of the sigma(70) subunit of RNA polymerase and the

transcriptional regulators Rsd from Escherichia coli and AlgQ from Pseudomonas

aeruginosa. J Bacteriol 183: 6413–6421.

50. Ouhammouch M, Adelman K, Harvey SR, Orsini G, Brody EN (1995)Bacteriophage T4 MotA and AsiA proteins suffice to direct Escherichia coli RNA

polymerase to initiate transcription at T4 middle promoters. Proc Natl Acad

Sci U S A 92: 1451–1455.51. Adelman K, Brody EN, Buckle M (1998) Stimulation of bacteriophage T4

middle transcription by the T4 proteins MotA and AsiA occurs at two distinctsteps in the transcription cycle. Proc Natl Acad Sci U S A 95: 15247–15252.

52. Hinton DM, March-Amegadzie R, Gerber JS, Sharma M (1996) Bacteriophage

T4 middle transcription system: T4-modified RNA polymerase; AsiA, a sigma70 binding protein; and transcriptional activator MotA. Methods Enzymol 274:

43–57.53. Hansen AM, Lehnherr H, Wang X, Mobley V, Jin DJ (2003) Escherichia coli SspA

is a transcription activator for bacteriophage P1 late genes. Mol Microbiol 48:1621–1631.

54. Pineda M, Gregory BD, Szczypinski B, Baxter KR, Hochschild A, et al. (2004) A

family of anti-sigma70 proteins in T4-type phages and bacteria that are similarto AsiA, a Transcription inhibitor and co-activator of bacteriophage T4. J Mol

Biol 344: 1183–1197.55. Sharma UK, Chatterji D (2008) Differential mechanisms of binding of anti-

sigma factors Escherichia coli Rsd and bacteriophage T4 AsiA to E. coli RNA

polymerase lead to diverse physiological consequences. J Bacteriol 190: 3434–3443.

56. Terry JM, Pina SE, Mattingly SJ (1991) Environmental conditions whichinfluence mucoid conversion Pseudomonas aeruginosa PAO1. Infect Immun 59:

471–477.

57. Testerman TL, Vazquez-Torres A, Xu Y, Jones-Carson J, Libby SJ, et al. (2002)The alternative sigma factor sigmaE controls antioxidant defences required for

Salmonella virulence and stationary-phase survival. Mol Microbiol 43: 771–782.58. Roychoudhury S, Sakai K, Chakrabarty AM (1992) AlgR2 is an ATP/GTP-

dependent protein kinase involved in alginate synthesis by Pseudomonas aeruginosa.Proc Natl Acad Sci U S A 89: 2659–2663.



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